Trypanosoma rangeli sialidase: cloning, expression and similarity to T. cruzi trans-sialidase.
Sialidases are hydrolytic enzymes present from virus to higher eukaryotes, catalyzing the removal of sialic acid from glycoconjugates. Some protozoa Trypanosomatidae secrete high levels of sialidase into the medium. We have now purified the secreted sialidase from Trypanosoma rangeli. Its N-terminal sequence reveals 100% identity with the corresponding region of the trans-sialidase from T. cruzi. Trans-sialidase, although homologous to viral and bacterial sialidases, displays a novel sialyltransferase activity and is involved in host cell invasion. Several homologous transsialidase-like genes were cloned from genomic DNA of T. rangeli, and grouped in three subfamilies. Active sialidase-encoding genes were found in one of them. The recombinant sialidase shows similar properties to those of the native enzyme, including undetectable trans-sialidase activity. Nevertheless, it has an overall identity of 68.9% with the catalytic domain of T. cruzi trans-sialidase, increasing to 86.7% admitting conservative substitutions. Only three other eukaryotic sialidases have been previously cloned, none of them showing significant homology to trans-sialidase. The isolation of a highly similar sialidase is relevant to further identify the molecular determinants allowing trans-sialidase activity. As a first approach, chimeric constructs between sialidase and trans-sialidase were generated, one of them rendering a sialidase with three times lower Km than the natural enzyme.
Buschiazzo
A
A
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, Argentina.
Campetella
O
O
Frasch
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D014345:Trypanosoma / Q000201:enzymology* / Q000235:genetics
D006023:Glycoproteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C069715:trans-sialidase; D009439:Neuraminidase